Prostate specific antigen (PSA) is a 34 kDa secretory protein of the human prostate (Wang, et ale, Invest. Urol., 17:159-163, 1979). The concentration of PSA in prostatic fluid and seminal plasma is very high, ranging from 0.5 to 2 mg/ml (Wang et al., Prostate, 2:89-96, 1981; Lilja et al., Prostate, 12:29-38, 1988). PSA is also present at elevated levels in serum of patients with prostate cancer. Because of this, serum PSA level has become an important tumor marker for the early detection and monitoring of patients with prostate cancer.
Serum PSA level is also elevated in patients with the non-cancerous disease, benign prostatic hyperplasia (BPH). Considerable studies have been performed in an effort to distinguish an elevated PSA serum level that is associated with prostate cancer from one that is associated with BPH. One area of study has focused on the various forms of PSA present in serum.
PSA has been shown to complex with protease inhibitors, such as .alpha..sub.1 -antichymotrypsin (ACT) and .alpha..sub.2 -macroglobulin (Christensson, et al., Eur. J. Biochem., 194:755-763, 1990). In serum, the majority of PSA is complexed with protease inhibitors, with only a small percentage of PSA remaining in its free form (Lilja, et ale, Clin. Chem., 37:1618-1625, 1991). Monitoring the relative amounts of the different forms of PSA (complexed versus free) in serum has been shown to be of potential clinical value in differentiating prostate cancer and BPH (Stenman et al., Cancer Res., 51:222-226, 1991; Christensson, et al., J. Urol., 150:100-105, 1993).
One way to detect the presence of PSA in a sample is by immunoassay. Immunoassays are assay systems that exploit the ability of an antibody to specifically recognize and bind to a particular target molecule. The region of a molecule that is recognized by an antibody, and to which the antibody binds is referred to as an "epitope." Large molecules, such as proteins, possess multiple epitopes. The molecule that is recognized by the antibody is also referred to as an "antigen."
Immunoassays are used extensively in modern diagnostics (Fackrell, J. Clin. Immunoassay 8:213-219, 1995). A large number of different immunoassay formats have been described (Yolken, R. H., Rev. Infect. Dis. 4:35, 1985); Ngo, T. T. et al., Enzyme Mediated Immunoassay, Plenum Press, N.Y., 1985). Immunoassay formats have been developed that are amenable to large scale usage.
The simplest immunoassay involves merely incubating an antibody that is capable of binding to a predetermined molecule (i.e. the "analyte") with a sample that is suspected to contain the analyte. The presence of the target molecule is determined by the presence, and is proportional to the concentration, of any immune complexes that form through the binding of antibody and analyte. In order to facilitate the separation of such immune complexes from the unbound antibody initially present, a solid phase is typically employed. In more sophisticated immunoassays, the concentration of the target molecule is determined by binding the antibody to a support, and then incubating the bound antibody in the presence of the analyte-containing sample.
Target molecules that have become bound to the immobilized antibody can be detected in any of a variety of ways. For example, the support can be incubated in the presence of a labeled, second antibody (i.e. a "sandwich" immunoassay) that is capable of binding to a second epitope of the target molecule. Immobilization of the labeled antibody on the support thus required the presence of the target, and is proportional to the concentration of the target in the sample. In an alternative assays, the sample is incubated with a known amount of labeled target and antibody binding site. Any target molecules present in the sample compete with the labeled target molecules for the antibody binding sites. Thus, the amount of labeled target molecules that are able to bind to the antibody is inversely proportional to the concentration of target molecule in the sample.
The various immunoassay formats can be further divided into two main classes, depending upon whether the assay requires the separation of bound species from unbound species. Heterogeneous immunoassays require such purification, and hence entail a separation or isolation step. Because homogeneous assays lack a separation step, and are more easily automated, they may be more desirable than heterogeneous assays in applications that entail the screening of large numbers of patients.
Regardless of immunoassay format, the utility of an immunoassay in detecting an analyte depends upon its capacity to report the extent of the formation of immune complexes between the antibody employed and the analyte whose presence or concentration is being measured. One approach for increasing this capacity involves labeling one or more the reagents.
A wide array of labels (such as radioisotopes, enzymes, fluorescent moieties, chemiluminescent moieties, or macroscopic labels, such as a bead, etc.) have been employed in order to facilitate the detection of immune complexes (Chard, T. et al., Laboratory Techniques and Biochemistry in Molecular Biology, North Holland Publishing Company, N.Y., 1978; Kemeny, D. M. et al., ELISA and Other Solid Phase Immunoassays, John Wiley & Sons, N.Y., 1988).
Immunodetection of the various forms of PSA in serum has become an important objective. Immunoassays to detect PSA in serum have been developed. These assays, however, measure all immunologically detectable forms of PSA in serum, i.e., PSA in its free form, in addition to PSA complexed with ACT. Immunoassays to measure only the free form of PSA have also been developed.
Prior immunoassays, however, have not been capable of selectively detecting the PSA-ACT complex. This is in part because antibodies that are specific for the PSA-ACT complex have not been developed. Because of the importance of detecting PSA, PSA-ACT and the relative amounts of each, it would be desirable to have an immunoassay capable of specifically detecting the PSA-ACT complex.
Previous attempts to generate antibodies specific for the PSA-ACT complex have been unsuccessful. Antibodies to PSA-ACT previously developed are not specific to PSA-ACT, but also bind free PSA, ACT or other serum ACT complexes, such as cathepsin G-ACT (CG-ACT). It is theorized that immunodominant sites on the PSA-ACT complex are not specific for the complex, but are also active for either PSA or ACT. What is needed are antibodies to PSA-ACT that do not significantly cross-react with PSA, ACT or CG-ACT.